Visible-light mediated selective phosphonylation modification of tryptophan residues in oligopeptides

Abstract

Despite their significant importance in biological and medicinal chemistry fields, the difficulties in the site-selective and diverse modification of biomolecules pose substantial obstacles to their applications. Here, we developed a direct C2–H phosphonylation strategy driven by visible light for specific modification of tryptophan containing peptides under exceedingly mild conditions, providing a straightforward and environmentally friendly synthetic method for the preparation of a plethora of phosphorylated tryptophan-containing peptides. Importantly, the protocol is applicable to the late-stage installation of phosphonate motifs into natural peptides, segetalin A and B, and their phosphonylation peptides exhibited better antiproliferative activity against HCT116 and HepG-2 compared with the original segetalins by a CCK-8 assay.

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Straightforward late-stage modification of biomolecules, which diversifies biomolecules in an elegant fashion without reliance on a cost- and time-intensive process compared with de novo peptide synthesis, bears enormous potential for chemical biology, proteomics, drug discovery, and pharmaceutical development.¹ Thus, the strategies for diversification and functionalization of biomolecules are highly appealing and have received a great deal of attention over the past decades.² As one of the proteinogenic amino acids, tryptophan (Trp) plays a crucial role in numerous biomolecules and exists in approximately 90% of native proteins despite its low relative abundance. The electron-rich π-system of the indole moiety from tryptophan makes it an excellent target for late-stage modification due to its diverse chemical reactivity. Past decades have witnessed the development of site- and chemoselective functionalization strategies toward the modification of Trp-containing peptides.³ For example, transition metal-catalyzed or metal-free catalyzed direct C2-selective functionalization of the indole ring of Trp or Trp-containing peptides, including arylation,⁴ fluoridation,⁵ alkylation,⁶ alkynylation,⁷ and so on, have been demonstrated (Scheme 1a). Moreover, photochemical methods have also increasingly been used for the selective modification of tryptophan residues in peptides in recent years by virtue of their distinct advantage to generate radicals in a mild and controlled fashion (Scheme 1b).⁸ Very recently, Sun and coworkers presented a DMSO/visible light co-mediated chemoselective modification of tryptophan residues in peptides through the construction of the C–S bond at the C2 position of the indole moiety.⁹ However, although major accomplishments have been achieved, most of the established strategies generally rely on highly active electrophiles and auxiliary directing groups to suppress the competitive reactions of other strongly nucleophilic species or more abundant residues due to their moderate nucleophilicity. Therefore, the development of distinctive chemical bioconjugation methods for the C2-selective functionalization of tryptophan with higher site selectivity and capability remains a necessity but a persistent challenge.

Scheme 1 C2-selective functionalization of tryptophan residues in peptides.

Phosphorus plays a pivotal role in regulating a wide range of cellular processes in life, and organophosphorus compounds have been investigated for their widespread occurrence in bioactive natural products, pharmaceuticals, agrochemicals etc. (Scheme 1c).¹⁰ In this context, the development of novel approaches for the selective phosphonylation modification of biomolecules is of prime importance. Unfortunately, however, in contrast to innumerable successes in the phosphonylation of simple aromatic heterocyclic compounds,¹¹ the phosphonylation of native Trp-based peptides remains scarce and not commonly used in synthesis due to the underlying difficulty in controlling site-selectivity in native protein peptides containing various residues, including –SH (Cys), –NH₂ (Lys) and/or –OH (Tyr). In this work, Trp's inherent photolability is profitably exploited, and a catalytic system driven by visible light that enables a direct C2–H phosphonylation strategy for specific modification of tryptophan-containing peptides under exceedingly mild conditions has therefore been developed, providing a straightforward and environmentally friendly synthetic method for the preparation of a plethora of phosphorylated tryptophan-containing peptides.

We began our studies by investigating the visible-light mediated selective phosphonylation of tryptophan with trimethyl phosphite (2) as the nucleophilic reagent (Table 1). After careful optimization of the reaction conditions, we found that the blue light irradiation of a mixture of methyl acetyl-L-tryptophanate (1) and trimethyl phosphite (2) in the presence of the photocatalyst [Ir(ppy)₂(dtbbpy)]PF₆ at 450–460 nm in MeCN under air for 18 h at room temperature gave the desired phosphonylation product 3 in 65% isolated yield (Table 1, entry 1). In the absence of light, this reaction could not proceed at all (entry 2). The results of screening wavelengths indicated that lamps operating within the blue light spectrum were all able to promote this phosphonylation transformation, and the optimum yield of the desired product 3 was obtained with the 450–460 nm light source (entries 3 and 4). It was also found that a photocatalyst was essential for this reaction (entry 5). In addition, Ir catalysts showed better activity compared with organic photocatalysts, and PC1 proved to be the optimal catalyst (entries 6–10). Alternative solvents were subsequently investigated, but no improvement was observed compared to the standard conditions (entries 11–16). Finally, we found that this photochemical transformation could not take place if the reaction system was purged with molecular nitrogen, which suggests that air was critical to this phosphonylation reaction (entry 17).

Table 1 Optimization of the reaction conditions^a

Entry	Deviation from standard conditions	Yield^b (%)
a Reactions were performed with 1 (0.2 mmol), 2 (1 mmol), and PC1 (3 mol%) in MeCN (2 mL) under irradiation at 450–460 nm using a 25 W LED lamp at room temperature (cooling by circulating water) for 18 h. b Isolated yield.
1	None	65
2	No light (dark)	nr
3	Irradiated with a 420–430 nm lamp	40
4	Irradiated with a 410–420 nm lamp	23
5	No PC	10
6	Catalyzed by PC2	55
7	Catalyzed by PC3	52
8	Catalyzed by PC4	30
9	Catalyzed by PC5	21
10	Catalyzed by PC6	15
11	Replaced MeCN with THF	61
12	Replaced MeCN with DMSO	39
13	Replaced MeCN with CH2Cl2	20
14	Replaced MeCN with isopropyl acetate	59
15	Replaced MeCN with toluene	nr
16	Replaced MeCN with H2O	nr
17	No air (N2)	nr

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With the optimal conditions established, we first investigated the feasibility of generalizing the visible light-mediated chemoselective phosphonylation modification of tryptophan and tryptophan-containing oligopeptides (Scheme 2). In addition to the model substrate methyl acetyl-L-tryptophanate (1), other protected tryptophans, such as ethyl (tert-butoxycarbonyl)-L-tryptophanate and methyl acetyl-L-tryptophanate, proceeded smoothly, giving the corresponding desired products (4–5) in moderate yields. In particular, unprotected amino acids, such as acetyl-L-tryptophan, (((9H-fluoren-9-yl)methoxy)carbonyl)-L-tryptophan and (tert-butoxycarbonyl)-L-tryptophan, could successfully be modified at the C-2 position of the indole moiety of tryptophan in moderate yields (6–8). Having realized the selective phosphonylation of simple tryptophan derivatives, we further employed a series of tryptophan-containing oligopeptides to evaluate the applicability. Encouragingly, under the standard conditions with phosphite ester as the coupling partner, a number of tryptophan-containing dipeptides that harbour other relatively inert residues such as phenylalanine, leucine, valine, and glutamate, were able to undergo this transformation, giving the corresponding phosphonylation dipeptides efficiently (9–16). It is worth noting that when a tryptophan-containing dipeptide with an electron-rich phenyl ring (S13) was used as the starting material, a trace amount of the phosphorylated side product occurring at the electron-rich arene moiety was observed by LCMS analysis. In addition, this protocol worked well with a variety of tripeptides, allowing for the selective phosphonylation of their tryptophan residues (17 and 18). It should be noted that Trp-containing oligopeptides bearing free –OH, –NH₂ and –SH groups, namely those containing unmasked lysine, tyrosine, serine and cysteine, could not deliver the desired products. Of them, Cys-containing peptides resulted in an unknown mixture, presumably due to the oxidation of sulfur atoms under these photoredox conditions.

Scheme 2 Tryptophan or oligopeptide (0.2 mmol), phosphite ester (1 mmol), and PC1 (3 mol%) in MeCN (2 mL) under irradiation at 450–460 nm using a 25 W LED lamp at room temperature (cooling by circulating water) for 18 h, isolated yield.

To further probe the reaction generality, we also examined complex peptide substrates (Scheme 3). Endomorphin, an endogenous peptide with potent analgesic activity owing to its remarkable selectivity and high affinity towards the μ-opioid receptor, could be transformed in good isolated yields under the optimized reaction conditions (20). Segetalin A and B, the natural peptides from Vaccaria segetalis, could also sustain phosphonylation modification exclusively at tryptophan residues in moderate isolated yields, ranging from 48 to 51% after purification by HPLC, which underscores the potential of this strategy for the late-stage modification of complex peptides (22–23, 25). Importantly, the obtained phosphonylation derivatives of segetalin were evaluated for their in vitro inhibitory activity toward HCT116 human colon cancer cells and HepG-2 human hepatocellular carcinoma cells, which showed better cell inhibitory activity compared with the original segetalins (see the ESI†).

Scheme 3 Late-stage functionalization of natural peptides.

To show the synthetic utility of this visible light-mediated chemoselective phosphonylation modification of tryptophan, a scale-up reaction was carried out with methyl acetyl-L-tryptophanate (1, 1 mmol) and trimethyl phosphite (2, 5 equiv.), delivering the desired product 3 in 54% isolated yield (Scheme 4).

Scheme 4 Scale-up reaction.

To gain deeper insight into the reaction mechanism, some control experiments were further executed. When radical scavengers, including 2,2,6,6-tetramethylpiperidinooxy (TEMPO), butylated hydroxytoluene (BHT) and 1,1-diphenylethylene (DPE), were added individually to the reaction of methyl acetyl-L-tryptophanate 1 with trimethyl phosphite 2 under the optimal conditions, a trace amount of the target product was observed in each case (Scheme 5A), and the coupling product of the indole radical intermediate from methyl acetyl-L-tryptophanate 1 with TEMPO or BHT was detected by HRMS (see Scheme S5†); these results suggest that the reaction might involve a radical intermediate. Cyclic voltammetry experiments showed that the oxidation potential of the excited photocatalyst [Ir(ppy)₂(dtbbpy)]PF₆ (E_1/2 (Ir^III*/Ir^II) = +0.66 V vs. SCE)¹² was lower than the oxidation potential of methyl acetyl-L-tryptophanate (1) (E_1/2 = +0.68 V vs. Ag/AgCl) or trimethyl phosphite (2) (E_1/2 = +0.71 V vs. Ag/AgCl) (see Scheme S9†). Accordingly, the reductive quenching pathway could be ruled out. The Stern–Volmer analysis also disclosed that the excited photocatalyst was quenched by oxygen other than acetyl-L-tryptophanate or trimethyl phosphite (see Scheme S6–S8†). Given that the energy transfer between the excited photocatalyst and oxygen is usual, singlet-state oxygen quenchers such as 1,4-diazabicyclo[2.2.2]octane (DABCO) and Co(acac)₃ were introduced into the standard reaction system, respectively. The results showed that these quenchers could completely quench the phosphonylation reaction. When anthracene 26 was added to this reaction, the trapping product of singlet oxygen was detected (Scheme 5B). Furthermore, tetraphenylporphyrin (TPP), a viable catalyst for singlet oxygen formation, proved to be beneficial for the visible-light mediated selective phosphonylation reaction (Scheme 5C). All of these results referred to the formation of singlet oxygen in the transformation.

Scheme 5 Control experiments.

Based on these observations and previous works,^11,13 a reasonable pathway is proposed, as shown in Scheme 6. Upon photoexcitation, PC produces an excited state PC*, which then interacts with dioxygen (O₂) by energy transfer to furnish singlet oxygen (¹O₂). Then, singlet oxygen (¹O₂) abstracts an electron from methyl acetyl-L-tryptophanate to give a superoxide radical and radical cation intermediate I, which is captured by P(OMe)₃ to generate intermediate II. The superoxide radical further performs a takeover of an electron from intermediate II to form the transient intermediate III and releases a hydroxyl anion (OH^–). Finally, the rearrangement of transient intermediate III gives the desired product.

Scheme 6 Plausible reaction pathway.

Conclusions

In conclusion, we have developed a facile and mild strategy for the phosphonylation modification of tryptophan-containing peptides under exceedingly mild conditions. This photoinduced site-selective process was applicable to the late-stage modification of complex peptides, such as segetalin A and B, and the obtained phosphorylated peptides exhibited better cell inhibitory activity toward HCT116 human colon cancer cells and HepG-2 human hepatocellular carcinoma cells compared with the original segetalins by a CCK-8 assay.

Author contributions

J. H., S. X., Y. L. and S. C. carried out the optimization and studied the scope of the reaction. J. L., P. C. and Y. Y. conducted the activity work. W. X., J. Z. and Z. C. directed the project. W. X. and J. Z. conceived the project and prepared the manuscript. All authors commented on this manuscript and gave approval to the final version of this manuscript.

Data availability

Electronic supplementary information (ESI) available: Experimental details, NMR spectra, and HRMS data. For the ESI† or other electronic format see https://doi.org/xxx.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

The authors thank the Discipline Construction Project of Guangdong Medical University (4SG24256G, 4SG23004G, and 4SG23249G), the Ordinary University Characteristic Innovation Project of Guangdong Province (2022KTSCX047) and the National Natural Science Foundation of China (22001075) for financial support.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d4qo01028k

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